Methods of blocking B-cell activation using anti-CD40 monoclonal antibodies

ABSTRACT

Methods for preventing or treating an antibody-mediated diease in a patient are presented, the methods comprising administration of a monoclonal antibody capable of binding to a human CD40 antigen located on the surface of a human B cell, wherein the binding of the antibody to the CD40 antigen prevents the growth or differentiation of the B cell. Monoclonal antibodies useful in these methods, and epitopes immunoreactive with such monoclonal antibodies are also presented.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/463,893, filedJun. 5, 1995, now U.S. Pat. No. 6,056,959, which is a divisionalapplication of U.S. Ser. No. 08/070,158, filed May. 28, 1993, now U.S.Pat. No. 5,677,165, which is a continuation-in-part of U.S. Ser. No.07/910,222, filed Jul. 9, 1992, now U.S. Pat. No. 5,397,703, currentlypending, the disclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to novel methods of treating diseases of theimmune system. In particular, this invention relates to methods ofpreventing or treating antibody-mediated diseases such as IgE-mediateddisease (allergies) and autoimmune diseases including sytemic lupuserythematosus (SLE), primary biliary cirrhosis (PBC), and idiopathicthrombocytopenic purpura (ITP).

BACKGROUND OF THE INVENTION

I. B-Cell Activation

B cells play an important role during the normal in vivo immuneresponse. A foreign antigen will bind to surface immunoglobulins onspecific B cells, triggering a chain of events including endocytosis,processing, presentation of processed peptides on MHC-class IImolecules, and up-regulation of the B7 antigen on the B-cell surface. Aspecific T cell then binds to the B cell via T-cell receptor (TCR)recognition of processed antigen presented on the MHC-class II molecule.Stimulation through the TCR begins to activate the T cell and initiatesT-cell cytokine production. Interaction between the CD28 antigen on Tcells and the B7 antigen on B cells can provide a second signal furtheractivating the T cell, resulting in high level cytokine secretion.Additionally, the CD40 ligand, which is not expressed on resting human Tcells, is up-regulated on the T-cell surface when the above-mentionedsignals are received. The B cell is then stimulated by the CD40 ligandthrough the CD40 antigen on the B-cell surface, and also by solublecytokines, causing the B cell to mature into a plasma cell secretinghigh levels of soluble immunoglobulin.

II. The EL4B5 Cell Line

A few years ago, Zubler et al., J. Immunol. (1985) 134:3662, observedthat a mutant subclone of the mouse thymoma EL-4 line, known as EL4B5,could strongly stimulate B cells of both murine and human origin toproliferate and differentiate into immunoglobulin-secreting plasma cellsin vitro. This activation was found to be antigen-independent and notMHC restricted. For optimal stimulation of human B cells, the presenceof supernatant from activated human T cells was needed, but a B-cellresponse also occurred when EL4B5 cells were preactivated withphorbol-12-myristate 13-acetate (PMA) or IL-1. Zubler et al.,Immnunological Reviews (1987) 99:281; and Zhang et al., J. Immunol.(1990) 144:2955. B-cell activation in this culture system isefficient—limiting dilution experiments have shown that the majority ofhuman B cells can be activated to proliferate and differentiate intoantibody-secreting cells. Wen et al. Eur. J. Immunol. (1987) 17:887.

The mechanism by which these mutant EL-4 cells activate both murine andhuman B cells has not been elucidated previously. It is, however, clearthat cell-cell contact is required for EL4B5-induced B-cell activation.First, B cells do not proliferate in the presence of supernatant fromPMA-stimulated EL4B5 cells. Zubler et al. (1985) supra. Second, B cellsdo not proliferate when they are separated from PMA-tated EL4B5 cells bya semipermeable filter membrane. Zhang et al., supra. Antibodies againstmouse LFA-1, human LFA-1 or human LFA-3 and antibodies against mouse orhuman MHC class II molecules do not inhibit EL4B5-induced proliferationof human or murine B cells. Zubler et al. (1987) and Zhang et al.,supra.

III. The CD40 Antigen the CD40 Antigen Ligand, and Anti-CD40 Antibodies

The CD40 antigen is a glycoprotein expressed on the cell surface of Bcells. During B-cell differentation the molecule is first expressed onpre-B cells and then disappears from the cell surface when the B cellbecomes a plasma cell. Crosslinking of the CD40 molecules with anti-CD40antibodies mediates a variety of effects on B cells. The CD40 antigen isknown to be related to the human nerve growth factor (NGF) receptor andtumor necrosis factor-alpha (TNF-α) receptor, suggesting that CD40 is areceptor for a ligand with important functions in B-cell activation.

A ligand for CD40 has been identified on the cell surface of activated Tcells. Fenslow et al., J. Immunol. (1992) 149:655; Lane et al., Eur. J.Immunol. (1992) 22:2573; Noelle et al., Proc. Natl. Acad. Sci. (USA)(1992) 89:6550. cDNA cloning of the CD40 ligand revealed a molecule withcharacteristics of a type-II transmembrane glycoprotein with homology toTNF-α. Armitage et al., Nature (1992) 357:80 and Spriggs et al., J. Exp.Med. (1992) 176:1543. The extracellular domain of the CD40 ligandcontains two arginine residues proximal to the transmembrane region,providing a potential proteolytic cleavage site that could give rise toa soluble form of the ligand. Expression of recombinant CD40 ligand hasdemonstrated that this molecule can stimulate the proliferation ofpurified B cells and, in combination with IL-4, mediate the secretion ofIgE. Armitage et al. and Spriggs et al., supra. It has been reportedthat abnormalities in the gene for the CD40 ligand, resulting in theabsence of a functional molecule on activated T cells, is responsiblefor the occurrence of X-linked hyper-IgM syndrome, a rare disordercharacterized by the inability of these patients to produce normallevels of antibody isotypes other than IgM. Allen et al., Science (1993)259:990; and Korthäuer et al., Nature (1993) 361:539.

All anti-CD40 antibodies known in the art have a stimulatory effect onhuman B cells. Cross-linking of the CD40 molecule on the B-cell surfaceusing known anti-CD40 antibodies mediates a variety of effects on Bcells. Anti-CD40 monoclonal antibodies (mAbs) can induce intercellularadhesion, proliferation and, in combination with certain cytokines,maturation to antibody secreting cells. For example, known anti-CD40mAbs have been shown to mimic the effects of T helper cells in B-cellactivation. When presented on adherent cells expressing FcγRII, theseantibodies induce Bell proliferation. J. Banchereau et al., Science(1989) 251:70. Moreover, the known anti-CD40 mAbs can replace the Thelper signal for secretion of IgM, IgG and IgE in the presence of IL-4.H. Gascan et al., J. Immunol. (1991) 147:8. Furthermore, known anti-CD40mAbs can prevent programmed cell death (apoptosis) of B cells isolatedfrom lymph nodes.

However, the anti-CD40 antibodies known in the art stimulate B cells butare incapable of inhibiting the B-cell response. Furthermore, noanti-CD40 antibodies are known that are (1) capable of inhibiting theB-cell response and (2) can be used to prevent or treatantibody-mediated disease.

SUMMARY OF THE INVENTION

The current invention is based on the discovery of anti-CD40 antibodiesthat do not stimulate the growth and differentiation of human B cells.In contrast, these antibodies can inhibit human B-cell responses atrelatively low concentrations. Accordingly, these antibodies can be usedto prevent or treat diseases or conditions that are mediated byantibodies produced by the human B-cell response. These antibodies alsorecognize novel epitopes on the CD40 antigen useful in modulating theB-cell response.

Accordingly, it is a primary object of this invention to provide amonoclonal antibody capable of binding to a human CD40 antigen locatedon the surface of a human B cell, wherein the binding of the antibody tothe CD40 antigen prevents the growth or differentiation of the B cell.

It is a further object of this invention to provide a method forpreventing or treating an antibody-mediate disease in a patient, themethod comprising administering to a patient in need of such treatment atherapeutically effective amount of a monoclonal antibody capable ofbinding to a human CD40 antigen located on the surface of a human Bcell, wherein the binding of the antibody to the CD40 antigen preventsthe growth or differentiation of the B cell, in a pharmaceuticallyacceptable excipient.

It is another object of this invention to provide a method forpreventing or treating an IgE-mediated disease such as an allergy in apatient, the method comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of a monoclonalantibody capable of binding to a human CD40 antigen located on thesurface of a human B cell, wherein the binding of the antibody to theCD40 antigen prevents the growth or differentiation of the B cell, in apharmaceutically acceptable excipient.

It is yet another object of this invention to provide a method forpreventing or treating an antibody-mediated autoimmune disease in apatient, the method comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of a monoclonalantibody capable of binding to a human CD40 antigen located on thesurface of a human B cell, wherein the binding of the antibody to theCD40 antigen prevents the growth or differentiation of the B cell, in apharmaceutically acceptable excipient. Particular autoimmune diseasescontemplated for treatment by this method include sytematic lupuserythematosus (SLE), primary biliary cirrhosis (PBC), and idiopathicthrombocytopenic purpura (ITP).

It is a further object of this invention to provide a CD40 antigenepitope capable of competing with the binding of a CD40 antigen to ananti-CD40 monoclonal antibody wherein the binding of that antibody to ahuman CD40 antigen located on the surface of a human B cell prevents thegrowth or differentiation of the B cell.

In more preferred embodiments of the above objects, the monoclonalantibody is either 5D12, 3A8 or 3C6.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic representation of the baculoviral transfervector pAcC8 and the sequence of the multiple cloning site (SEQ ID NO:1). As shown, the multiple cloning site was inserted between nucleotidenumber +37 and +176 of the polyhedrin gene. FIG. 1B shows a schematicrepresentation of the generation of Sf9 cells which express human CD40or B7 antigen.

FIG. 2 shows the sequences of polymerase chain reaction primers used inthe preparation of coding regions for human CD40 and human B7 antigens.These primers were constructed on the basis of the published completeDNA coding sequences for antigens B7 and CD40. Specifically shown arethe primers for full length B7, which are designated as MR67 (SEQ ID NO:2) and MR69 (SEQ ID NO: 3); the primers for soluble B7, which aredesignated as MR67 (SEQ ID NO: 2) and MR145 (SEQ ID NO: 4); the primersfor full length CD40, which are designated as MR108 (SEQ ID NO: 5) andMR112 (SEQ ID NO: 6); and the primers for soluble CD40, which aredesignated as MR108 (SEQ ID NO: 5) and MR150 (SEQ ID NO: 7).

FIG. 3 shows the results of ELISA assays examining the reaction ofanti-CD40 monoclonal antibody 52C6 with Sf9 cells expressing CD40 andwith Sf9 cells expressing B7.

FIGS. 4A-4C shows the results of the fluorescent cell staining ofEBV-transformed B-cell line ARC cells expressing CD40. FIG. 4A shows theresults of staining ARC EBV transformed cells with serum from a mouseimmunized with B7 expressing Sf9 cells (solid line) or with normal mouseserum (dotted line). FIG. 4B shows the results of staining ARC EBVtransformed cells with serum from a mouse immunized with CD40 expressingSf9 cells (solid line) or with normal mouse serum (dotted line). FIG. 4Cshows the results of staining ARC EBV transformed cells with serum froma mouse imunized with control Sf9 cells (solid line) or with normalmouse serum (dotted line).

FIG. 5A compares the ability of three new and one old anti-CD40 mAbs toco-stimulate anti-IgM induced human B-cell proliferation. FIG. 5Brepeats the experiment of FIG. 5A in the presence of recombinantinterleukin-2 (rIL-2).

FIG. 6 shows the ability of three new anti-CD40 mAbs to inhibit humanB-cell proliferation induced by costimulation with immobilized anti-IgMand anti-CD40 mAb 52C6.

FIG. 7 shows the effect of three new anti-CD40 mAbs on EL4B5-inducedhuman B-cell proliferation.

FIG. 8 shows the effect of soluble CD40 (hCD40. μ) on EL4B5-inducedhuman B-cell proliferation.

FIGS. 9A and 9B show the effect of one new anti-CD40 mAb 5D12 on humanT-cell induced immunoglobulin production by human B cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein draws on previously published work andpending patent applications. By way of example, such work consists ofscientific papers, patents or pending patent applications. All of thesepublications and applications, cited previously or below are herebyincorporated by reference.

Definitions

As used herein, the term “antibody” refers to polyclonal antibodies,monoclonal antibodies, humanized antibodies, single-chain antibodies,and fragments thereof such as F_(ab), F_((ab′)2), F_(v), and otherfragments which retain the antigen binding function of the parentantibody.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins as well as fragments such as F_(ab),F_((ab′)2), F_(v), and others which retain the antigen binding functionof the antibody. Monoclonal antibodies of any mammalian species can beused in this invention. In practice, however, the antibodies willtypically be of rat or murine origin because of the availability of rator murine cell lines for use in making the required hybrid cell lines orhybridomas to produce monoclonal antibodies.

As used herein, the term “humanized antibodies” means that at least aportion of the framework regions of an immunoglobulin are derived fromhuman immunoglobulin sequences.

As used herein, the term “single chain antibodies” refer to antibodiesprepared by determining the binding domains (both heavy and lightchains) of a binding antibody, and supplying a linking moiety whichpermits preservation of the binding function. This forms, in essence, aradically abbreviated antibody, having only that part of the variabledomain necessary for binding to the antigen. Determination andconstruction of single chain antibodies are described in U.S. Pat. No.4,946,778 to Ladner et al.

The term “CD40 antigen epitope” as used herein refers to a moleculewhich is capable of immunoreactivity with the anti-CD40 monoclonalantibodies of this invention, excluding the CD40 antigen itself. CD40antigen epitopes may comprise proteins, protein fragments, peptides,carbohydrates, lipids, and other molecules, but for the purposes of thepresent invention are most commonly proteins, short oligopeptides,oligopeptide mimics (i.e., organic compounds which mimic the antibodybinding properties of the CD40 antigen), or combinations thereof.Suitable oligopeptide mimics are described, inter alia, in PCTapplication US91/04282.

The Antibody

The antibodies of the current invention bind to a human CD40 antigen onthe surface of a human B cell and do not stimulate the growth ordifferentiation of the B cell. These antibodies may be polyclonalantibodies, monoclonal antibodies, humanize antibodies, single-chainantibodies, and fragments thereof.

1. Antibody Preparation

Monoclonal antibodies 5D12, 3A8 and 3C6 are prepared as described inExample 1 herein. Other monoclonal antibodies of the invention may beprepared similarly, or as follows. First, polyclonal antibodies areraised against the CD40 antigen. Second, monoclonal antibodies specificfor CD40 are selected.

a) Polyclonal Sera

Polyclonal sera may be prepared by conventional methods. In general, asolution containing the CD40 antigen is first used to immunize asuitable animal, preferably a mouse, rat, rabbit or goat. Rabbits andgoats are preferred for the preparation of polyclonal sera due to thevolume of serum obtainable, and the availability of labeled anti-rabbitand anti-goat antibodies. Immunization is generally performed by mixingor emulsifying the antigen-conttig solution in saline, preferably in anadjuvant such as Freund's complete adjuvant, and injecting the mixtureor emulsion parenteally (generally subcutaneously or intramuscularly). Adose of 50-200 μg/injection is typically sufficient. Immuniztion isgenerally boosted 2-6 weeks later with one or more injections of theprotein in saline, preferably using Freund's incomplete adjuvant. Onemay alternatively generate antibodies by in vitro immunization usingmethods known in the art, which for the purposes of this invention isconsidered equivalent to in vivo immunization.

Polyclonal antisera are obtained by bleeding the immunized animal into aglass or plastic container, incubating the blood at 25° C. for one hour,followed by incubating at 4° C. for 2-18 hours. The serum is recoveredby centrifugation (e.g., 1,000×g for 10 minutes). About 20-50 ml perbleed may be obtained from rabbits.

b) Monoclonal Antibodies

Monoclonal antibodies are prepared using the method of Kohler andMilstein, Nature (1975) 256:495-96, or a modification thereof.Typically, a mouse or rat is immunized as described above. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) are removed and dissociated intosingle cells. If desired, the spleen cells may be screened (afterremoval of nonspecifically adherent cells) by applying a cell suspensionto a plate or well coated with the protein antigen. B-cells expressingmembrane-bound immunoglobulin specific for the antigen bind to theplate, and are not rinsed away with the rest of the suspension.Resulting B-cells, or all dissociated spleen cells, are then induced tofuse with myeloma cells to form hybridomas, and are cultured in aselective medium (e.g., hypoxanthine, aminopterin, thymidine medium,“HAT”). The resulting hybridomas are plated by limiting dilution, andare assayed for the production of antibodies which bind specifically tothe desired immunizing cell-surface antigen (and which do not bind tounrelated antigens). The selected mAb-secreting hybridomas are thencultured either in vitro (e.g., in tissue culture bottles or hollowfiber reactors), or in vivo (as ascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may belabeled using conventional techniques. Suitable labels includefluorophores, chromophores, radioactive atoms (particularly ³²P and¹²⁵I), electron-dense reagents, enzymes, and ligands having specificbinding partners. Enzymes are typically detected by their activity. Forexample, horseradish peroxidase is usually detected by its ability toconvert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment,quantifiable with a spectrophotometer. “Specific binding partner” refersto a protein capable of binding a ligand molecule with high specificity,as for example in the case of an antigen and a monoclonal antibodyspecific therefor. Other specific binding partners include biotin andavidin or streptavidin, IgG and protein A, and the numerousreceptor-ligand couples known in the art. It should be understood thatthe above description is not meant to categorize the various labels intodistinct classes, as the same label may serve in several differentmodes. For example, ¹²⁵I may serve as a radioactive label or as anelectron-dense reagent. HRP may serve as enzyme or as antigen for a mAb.Further, one may combine various labels for desired effect. Forexamnple, mAbs and avidin also require labels in the practice of thisinvention: thus, one might label a mAb with biotin, and detect itspresence with avidin labeled with ¹²⁵I, or with an anti-biotin mAblabeled with HRP. Other permutations and possibilities will be readilyapparent to those of ordinary skill in the art, and are considered asequivalents within the scope of the instant invention.

CD40 Antigen Epitopes

The CD40 antigen epitopes of this invention are molecules that areimmunorccctive with anti-CD40 monoclonal antibodies whose binding to ahuman CD40 antigen located on the surface of a human B cell prevents thegrowth or differentiation of the B cell. That is, such epitopes competewith the binding of said antibodies to the CD40 antigen. Systematictechniques for identifying these epiotpes are known in the art, asdescribed by H. M. Geysen in U.S. Pat. No. 4,708,871, which is incorpodherein by reference. Typically these epitopes are short amino acidsequences. These sequences may be embedded in the sequence of longerpeptides or proteins, as long as they are accessible.

The epitopes of the invention may be prepared by standard peptidesynthesis techniques, such as solid-phase synthesis. Alternatively, thesequences of the invention may be incorporated into larger peptides orproteins by recombinant methods. This is most easily accomplished bypreparing a DNA cassette which encodes the sequence of interest, andligating the cassette into DNA encoding the protein to be modified atthe appropriate site. The sequence DNA may be synthesized by standardsynthetic techniques, or may be excised from the phage pIII gene usingthe appropriate restriction enzymes.

Epitopes identified herein may be prepared by simple solid-phasetechniques. The minimum binding sequence may be determinedsystematically for each epitope by standard methods, for example,employing the method described by H. M. Geysen, U.S. Pat. No. 4,708,871.Briefly, one may synthesize a set of overlapping oligopeptides derivedfrom the CD40 antigen bound to a solid phase array of pins, with aunique oligopeptide on each pin. The pins are araanged to match theformat of a 96-well micmoiter plate, permitting one to assay all pinssimultaneously, e.g., for binding to an anti-CD40 monoclonal antibody.Using this method, one may readily determine the binding affinity forevery possible subset of consecutive amino acids.

Analogs of the invention are also prepared by standard solid-phasemethods, and those methods described in PCT application US91/04282.

Formulations and Methods of Administration

The antibodies of this invention are administered at a concentrationthat is therapeutically effective to prevent or treat antibody-mediateddiseases such as allergies, SLE, PBC and ITP. To accomplish this goal,the antibodies may be formulated using a variety of acceptableexcipients known in the art. Typically, the antibodies are administeredby injection, either intravenously or intraperitoneally. Methods toaccomplish this administration are known to those of ordinary skill inthe art. It may also be possible to obtain compositions which may betopically or orally administered, or which may be capable oftransmission across mucous membranes.

Before administration to patients, formulants may be added to theantibodies. A liquid formulation is preferred. For example, theseformulants may include oils, polymers, vitamins, carbohydrates, aminoacids, salts, buffers, albumin, surfactants, or bulking agents.Preferably carbohydrates include sugar or sugar alcohols such as mono,di, or polysaccharides, or water soluble glucans. The saccharides orglucans can include fructose, dextrose, lactose, glucose, mannose,sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha andbeta cyclodextrin, soluble starch, hydroxethyl starch andcarboxymethylcellulose, or mixtures thereof. Sucrose is most preferred.“Sugar alcohol” is defined as a C₄ to C₈ hydrocarbon having an —OH groupand includes galactitol, inositol, mannitol, xylitol, sorbitol,glycerol, and arabitol. Mannitol is most preferred. These sugars orsugar alcohols mentioned above may be used individually or incombination. There is no fixed limit to amount used as long as the sugaror sugar alcohol is soluble in the aqueous preparation. Preferably, thesugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %,more preferable between 2.0 and 6.0 w/v %. Preferably amino acidsinclude levorotary (L) forms of carnitine, arginine, and betaine;however, other amino acids may be added. Preferred polymers includepolyvinylpyrrolidone (PVP) with an average molecular weight between2,000 and 3,000, or polyethylene glycol (PEG) with an average molecularweight between 3,000 and 5,000. It is also preferred to use a buffer inthe composition to minimize pH changes in the solution beforelyophilization or after reconstitution. Most any physiological buffermay be used, but citrate, phosphate, succinate, and glutamate buffers ormixtures thereof are preferred. Most preferred is a citrate buffer.Preferably, the concentration is from 0.01 to 0.3 molar. Surfactantsthat can be added to the formulation are shown in EP Nos. 270,799 and268,110.

Additionally, antibodies can be chemically modified by covalentconjugation to a polymer to increase their circulating half-life, forexample. Preferred polymers, and methods to attach them to peptides, areshown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546which are all hereby incorporated by reference in their entireties.Preferred polymers are polyoxyethylated polyols and polyethylene glycol(PEG). PEG is soluble in water at room temperature and has the generalformula: R(O—CH₂—CH₂)_(n)O—R where R can be hydrogen, or a protectivegroup such as an alkyl or alkanol group. Preferably, the protectivegroup has between 1 and 8 carbons, more preferably it is methyl. Thesymbol n is a positive integer, preferably between 1 and 1,000, morepreferably between 2 and 500. The PEG has a preferred average molecularweight between 1000 and 40,000, more preferably between 2000 and 20,000,most preferably between 3,000 and 12,000. Preferably, PEG has at leastone hydroxy group, more preferably it is a terminal hydroxy group. It isthis hydroxy group which is preferably activated to react with a freeamino group on the inhibitor. However, it will be understood that thetype and amount of the reactive groups may be varied to achieve acovalently conjugated PEG/antibody of the present invention.

Water soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), etc. POG is preferred. Onereason is because the glycerol backbone of polyoxyethylated glycerol isthe same backbone occurring naturally in, for example, animals andhumans in mono-, di-, triglycerides. Therefore, this branching would notnecessarily be seen as a foreign agent in the body. The POG has apreferred molecular weight in the same range as PEG. The structure forPOG is shown in Knauf et al., 1988, J. Bio. Chem. 263:15064-15070, and adiscussion of POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106,both of which are hereby incorporated by reference in their entireties.

Another drug delivery system for increasing circulatory half-life is theliposome. Methods of preparing liposome delivery systems are discussedin Gabizon et al., Cancer Research (1982) 42:4734; Cafiso, BiochemBiophys Acta (1981) 649:129; and Szoka, Ann Rev Biophys Eng (1980)9:467. Other drug delivery systems are known in the art and aredescribed in , e.g., Poznansky et al., DRUG DELIVERY SYSTEMS (R. L.Juliano, ed., Oxford, N.Y. 1980), pp. 253-315; M. L. Poznansky, PharmRevs (1984) 36:277.

After the liquid pharmaceutical composition is prepared, it ispreferably lyophilized to prevent degradation and to preserve sterility.Methods for lyophilizing liquid compositions are known to those ofordinary skill in the art. Just prior to use, the composition may bereconstituted with a sterile diluent (Ringer's solution, distilledwater, or sterile saline, for example) which may include additionalingredients. Upon reconstitution, the composition is preferablyadministered to subjects using those methods that are known to thoseskilled in the art.

As stated above, the antibodies and compositions of this invention areused to treat human patients to prevent or treat antibody-mediateddiseases such as allergies, SLE, PBC and ITP. The preferred route ofadministration is parenteraly. In parenteral administration, thecompositions of this invention will be formulated in a unit dosageinjectable form such as a solution, suspension or emulsion, inassociation with a pharmaceutically acceptable parenteral vehicle. Suchvehicles are inherently nontoxic and nontherapeutic. Examples of suchvehicles are saline, Ringer's solution, dextrose solution, and Hanks'solution. Nonaqueous vehicles such as fixed oils and ethyl oleate mayalso be used. A preferred vehicle is 5% dextrose in saline. The vehiclemay contain minor amounts of additives such as substances that enhanceisotonicity and chemical stability, including buffers and preservatives.

The dosage and mode of administration will depend on the individual.Generally, the compositions are administered so that antibodies aregiven at a dose between 1 μg/kg and 20 mg/kg, more preferably between 20μg/kg and 10 mg/kg, most preferably between 1 and 7 mg/kg. Preferably,it is given as a bolus dose, to increase circulating levels by 10-20fold and for 4-6 hours after the bolus dose. Continuous infusion mayalso be used after the bolus dose. If so, the antibodies may be infusedat a dose between 5 and 20 μg/kg/minute, more preferably between 7 and15 μg/kg/minute.

The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and are not to be construed as restricting the invention inany way.

EXAMPLES

Materials and Methods

Cell Lines

The mutant mouse thymoma EL-4 subclone EL4B5 was a gift of Dr. R. H.Zubler, Hôpital Cantonal Universitaire, Geneva. Mouse 3T6 transfectantcells expressing hybrid molecules of the HR (high responder) allelicform of human FcγRIIa were a gift of Dr. P. A. M. Warmerdam, Departmentof Experimental Immunology, University Hospital Utrecht, Utrecht, TheNetherlands. Warmerdam et al., J. Immunol. (1991) 147:1338. Both celllines were cultured in Iscove's Modified Dulbecco's Medium (IMDM),supplemented with gentamycin (80 μg/ml) and 10% heat-inactivated fetalcalf serum (FCS) (Hyclone, Logan, Utah). To avoid possible loss of Bcell activating capacity, every 4 to 8 weeks a new batch of EL4B5 cellswas thawed. The cell lines were periodically tested for mycoplasmacontamination by the use of a ³H-labelled DNA probe for mycoplasmaribosomal RNA (GenProbe, San Diego, Calif.) and were free of mycoplasmaduring the course of the experiments.

Antibodies and hCD40.Hμ Fusion Protein

Anti-CD40 mAb 5D12, 3C6 and 3A8 were generated by immunizing mice withinsect cells expressing recombinant human CD40 as shown in Example 1.Anti-(B7) mAb B7-24 was generated in a similar way by immunizing withinsect cells expressing recombinant human B7. Anti-CD40 mAb S2C6 was agift of Dr. S. Paulie (University of Stockholm, Sweden). Paulie et al.,J. Immunol. (1989) 142:590. Anti-CD40 mAb G28.5 was donated by Dr. J. A.Ledbetter (Oncogen Corporation, Seattle, Wash., USA). Clark et al., PNAS(USA) (1986) 83:4494. Control antibodies were:anti-(β-glucocerebrosidase) mAb 8E4 (IgG1), Barneveld et al., Eur. J.Biochem. (1983) 134:585, and myeloma immunoglobulins MOPC-21 (IgG1) andMOPC-141 (IgG2b) (Sigma, St. Louis, Mo.). All mAb were used as purifiedantibody preparations. hCD40.Hμ fusion protein was a gift of Dr. P. Lane(Basel Institute for Immunology, Basel, Switzerland) and was used as a5× concentrated supernatant of transfected J558L cells. Lane et al.,Eur. J. Immunol. (1992) 22:2573.

Human B Lymphocytes

B lymphocytes were isolated from tonsils obtained from childrenundergoing tonsillectomies, essentially as described in De Groot et al.,Lymphokine Research (1990) 9:321. Briefly, the tissue was dispersed withscalpel blades, phagocytic and NK cells were depleted by treatment with5 mM L-leucine methyl ester and T cells were removed by one cycle ofresetting with sheep erythrocytes (SRBC) treated with 2-aminoethylisothiouronium bromide. The purity of the resulting B lymphocytepreparations was checked by indirect immunofluorescent labelling withanti-(CD20) mAb B1 (Coulter Clone, Hialeah, FA) or anti-(CD3) mAb OKT3(Ortho, Raritan, N.J.) and a FITC-conjugated F(ab′)₂ fragment of rabbitanti-(mouse Ig) (Zymed, San Francisco, Calif.), and FACS analysis. The Bcell preparations contained (mean±SD of 6 isolations): 95±4%CD20-positive cells and 2±1% CD3-positive cells.

B-Cell Proliferation Assay

B cells (4×10⁴ per well) were cultured in 200 μl IMDM supplemented with10% fetal calf serum in flat bottom 96-well microtiter plates. B cellswere stimulated by addition of immobilized anti-(IgM) antibodies(Immunobeads; 5 μg/ml; BioRad, Richmond, Calif.). Where indicated 100U/ml recombinant IL-2 was added. Varying concentrations of mAbs wereadded at the onset of the microcultures and proliferation was assessedat day 3 by measurement of the incorporation of [³H]-thymidine after 18hour pulsing.

Banchereau-Like B-Cell Proliferation Assay

For testing the ability of anti-CD40 mAbs to stimulate B-cellproliferation in a culture system analogous to that described byBanchereau et al., Science (1991) 251:70, mouse 3T6 transfectant cellsexpressing the HR allellic form of human FcγRII were used. B cells(2×10⁴ per well) were cultured in flat-bottom microwells in the presenceof 1×10⁴ transfectant cells (irradiated with 5000 Rad) in 200 μl IMDMsupplemented with 10% fetal calf serum and 100 U/ml recombinant IL-4.Before addition of the B cells, the 3T6 cells were allowed to adhere tothe culture plastic for at least 5 hours. Anti-CD40 mAbs were added atconcentrations varying from 15 ng/ml to 2000 ng/ml and proliferation ofB cells was assessed by measurement of thymidine incorporation at day 7,upon 18 hour pulsing with [³H]-thymidine.

B-Cell Activation Assay with EL4B5 Cells

B cells (1000 per well) were cultured together with irradiated (5000Rad) EL4B5 cells (5×10⁴ per well) in flat bottom microtiter plates in200 μl IMDM supplemented with 10% heat-inactivated fetal calf serum, 5ng/ml phorbol-12-myristate 13-acetate (Sigma) and 5% human T-cellsupernatant. MAbs were added at varying concentrations at the onset ofthe cultures and thymidine incorporation was assessed at day 6 after 18hour pulsing with [³H]-thymidine. For the preparation of T-cellsupernatant, purified T cells were cultured at a density of 10⁶/ml for36 hours in the presence of 1 μg/ml PHA and 10 ng/ml PMA. Wen et al.,supra. T-cell supernatant was obtained by centrifugation of the cellsand stored at −20° C. The effectiveness of T-cell supernatants inenhancing proliferation of human B cells in EL4B5-B cell cultures wastested and the most effective supernatants were pooled and used in theexperiments.

Human T Cell Helper Assay for Antibody Production by B Cells

96-well tissue culture plates were coated with a 1:500 dilution ofascites fluid of anti-CD3 mAb CLB-T3/3 (CLB, Amsterdam, TheNetherlands). As indicated costimulatory mAbs were added: anti CD2 mAbsCLB-T11.1/1 and CLB-T11.2/1 (CLB, Amsterdam, The Netherlands), bothascites 1:1000 and anti-CD28 mAb CLB-28/1 (CLB, Amsterdam, TheNetherlands). Subsequently, tonsillar T cells (irradiated, 3000 Rad; 10⁵per well), tonsillar B cells (10⁴ per well) and rIL-2 (20 U/ml) wereadded. The final volume of each cell culture was 200 μl. After 8 days,cells were spun down, and cell-free supernatant was harvested. Theconcentrations of human IgM and IgG in (diluted) samples were estimatedby ELISA as described below.

ELISA Assay for Immunoglobulin Quantification

The concentrations of human IgM and IgG were estimated by ELISA. 96-wellELISA plates were coated with 4 μg/ml mouse anti-human IgG mAb MH 16-01(CLB, Amsterdam, The Netherlands) or with 1.2 μg/ml mouse anti-human IgMmAb 4102 (Tago, Burlingame, Calif.) in 0.05 M carbonate buffer (pH=9.6),by incubation for 16 h at 4° C. Plates were washed 3 times withPBS-0.05% Tween-20 (PBS-Tween) and saturated with BSA for 1 hour. After2 washes the plates were incubated for 1 h at 37° C. with differentdilutions of the test samples. After 3 washes, bound Ig was detected byincubation for 1 h at 37° C. with 1 μg/ml peroxidase-labeled mouseanti-human IgG mAb MH 16-01 (CLB) or mouse anti-human IgM mAb MH 15-01(CLB). Plates were washed 4 times and bound peroxidase activity wasrevealed by the addition of O-phenylenediamine as a substrate. Humanstandard serum (H00, CLB) was used to establish a standard curve foreach assay.

Flow Cytofluorometric Assay

ARC cells (10⁶ cells/sample) were incubated in 100 μl primary antibody(10 μg/ml in PBS-BSA or Hankis' balanced salt solution (HBSS)supplemented with 1% BSA and 0.05% sodium azide) for 20 min at 4° C.After 3 washes with PBS-BSA or HBSS-BSA, the cells were incubated in 100μl FITC-labeled F(ab′)₂ fragments of goat anti-(mouse IgG) antibodies(Jackson, West Grove, Pa.) for 20 min at 4° C. After 3 washes withPBS-BSA or HBSS-BSA and 1 wash with PBS, the cells were resuspended in0.5 ml PBS. Analyses were performed with a FACSCAN V (Becton Dickinson,San Jose, Calif.).

Alternatively, EL4B5 cells were harvested before and at different timepoints during culture in medium containing PMA (5ng/ml) and human T-cellsupernatant (5%). Cells were incubated for 30 minutes with 10 μlsupernatant of transfected cells contning hCD40-Hμ diluted in 100 μlHank's Balanced Salt Solution supplemented with 0.05% sodium azide (4°C.). This was followed by incubation with FITC-conjugated F(ab′)₂fragments of rabbit anti-(human IgM) (Central Laboratory of the BloodTransfusion Service, Amsterdam, The Netherlands). As a control, cellswere incubated with the FIT-conjugate only. For analysis a FACScan-4cytofluorometer (Becton and Dickinson) was used. Non-vital cells wereexcluded from analysis by the use of propidium iodide.

Example 1 Making Monoclonal Antibodies to B7 and CD40

A. PCR Cloning of CD40 and B7

RNA was isolated from a population of EBV-tmnsformed human spleen cellsessentially as described by Chirgwin et al., Biochemistry (1979)17:5294. In brief, the cells were washed twice with phosphate bufferedsaline (PBS) and lysed in 5 M guanidinium thiocyanate in the presence of0.7 M 2-mercaptoethanol. The cell lysate was layered on a discontinuousCsCl gradient (Chirgwin et al.) and centrifuged for 16 hours at 26,000rpm in a Beckman SW28 rotor. The RNA was recovered by dissolving thepellet in DEPC-treated H₂O. The RNA was precipitated with ethanol once,resuspended in DEPC-treated H₂O, and stored at −70° C.

Total RNA (10 μg/reaction) was converted to cDNA using random hexamerpriming in 50 μl reaction buffer containing 500 units MLV-RT (BethesdaResearch Laboratories, Bethesda, Md.), 5 μM random hexamer (Pharmacia,Piscataway, N.J.), 1 mM DTT, dNTP mix (0.5 mM each), 10 mM Tris-HCL pH8.3, 50 mM KCl, 2.5 mM MgCl₂ and 0.1 mg/ml BSA (bovine serum albumin).After incubation at 37° C. for 1 hour, the samples were boiled for 3minutes and stored at −70° C. The DNA encoding the CD40 and B7 moleculeswas generated by PCR using primers which contained sequences havinghomology to known CD40 and B7 sequence, where the primers also encodedrestriction sites useful for cloning (FIG. 2). These primers were basedon the published cDNA coding sequences for B7 and CD40. Freeman et al.,J. Immunol. (1989) 143:2714, and Stamenkovic et al., EMBO J. (1989)8:1403. All primers start with a C-G clamp at the 5′ end followed by arestriction site for cloning (shown in bold, FIG. 2). The underlinedsequences in the backward primers, for the cloning of the soluble formsof B7 and CD40, represents an epitope recognized by a monoclonalantibody used for affinty purification. The numbers in bracketsrepresent the location of the primers relative to the published cDNAsfor CD40 and B7.

For PCR amplification, 1 μl of cDNA was mixed with 1 μl (10 picomoles)of a forward primer, 1 μl (10 picomoles) of a backward primer, and 47 μlof PCR mix. The PCR mix consisted of 1.25 units Taq polymerase(Perkin-Elmer/Cetus, Norwalk, Conn.), dNTP mix (0.2 mM each), 10 mMTris-HC 1 pH 8.3, 50 mM KCl, 2.5 mM MgCl₂ and 0.1 mg/ml BSA. The 50 μlof PCR mixture was overlaid with 70 μl mineral oil and subjected to 25cycles of amplification in a Perkin-Elmer/Cetus thermocycler(denaturation at 95° C. for 30 seconds, primer annealing at 55° C. for30 seconds and extension at 72° C. for 1.5 minutes). PCR products wereobtained after 25 amplification cycles.

The amplification products were digested with BglII and KpnI (FIG. 1B)and isolated by size-fractionation. Before expression in baculovirus,the DNA sequence of each fragment was confirmed by sequencing analysisto prevent the introduction of PCR-induced mutations. The baculovirustransfer vector pAcC8 was also digested with BglII and KpnI (FIG. 1B).

The amplified flagments were ligated to the linear pAcC8 vector (ratioof insert to vector was 3:1). The ligation products were transformedinto bacterial strain DH5α (Gibco/BRL, Gaithersburg Md.) and recombinantpAcC8 vectors were selected on the basis of ampicillin resistance.Recombinant plasmids were isolated from bacteria clones (Maniatis etal., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratories), 1982; Ausubel et al., CurrentProtocols in Molecular Biology (Media, PA: John Wiley and Sons)) and thepresence of the insert of interest verified using polymerase chainreactions (see above). Large scale plasmid preparation was performed bystandard procedures (Ausubel et al.; Maniatis et al; Sambrook et al.,Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratories), 1989).

B. Baculovirus Expression of Human CD40 and B7

Sequences encoding human CD40 and human B7 were recombined into theAutographa californica baculovirus (AcNPV) using the transfer vectorspAcCD40 (encoding the full-length CD40 molecule), pAcCD40-ED/Glu(encoding the extracellular domain of CD40), pAcB7 (encoding thefull-length B7 molecule) and pCcB7-ED/Glu (encoding the cellular domainof the B7 molecule).

The plasmids were cotransfected with wild-type baculoviral DNA (2-10pfu) (AcNPV; Summers et al., A Manual of Methods for Baculovirus Vectorsand Insect Cell Culture Procedures, Texas Agricultural ExperimentalStation Bulletin No. 1555 (1987)) into Sf9 (Spodoptera frugiperda) cellsat a density of 10⁶ cells/ml (Summers et al.). Recombinantbaculovirus-infected Sf9 cells were identified and clonally purified(Summers et al.).

For cell surface expression of recombinant proteins the cells wereharvested after 48 hours of culture.

C. Sf9 Insect Cell ELISA

Sf9 insect cells infected with recombinant virus were cultured for 48hours in 24-well plates. After removal of the tissue culture medium theplates were incubated for 45 minutes at room temperature (RT) with 0.25ml of antibody in PBS with 1% BSA (PBS-BSA). After three washed withPBS-BSA, the plates were incubated for 35 minutes at RT with 250 μl of a1/250 dilution of goat anti-(mouse total Ig) immunoglobulins conjugatedto horseradish peroxidase (Zymed, South San Francisco, Calif.) inPBS-BSA. Unbound peroxidase activity was removed by washing five timeswith PBS-BSA. Bound peroxidase activity was revealed by the addition ofan assay mixture prepared by diluting 0.5 ml of 2 mg/ml3,3′,5,5′-tetramethylbenzidine in ethanol to 10 ml with 10 mM sodiumacetate, 10 mM EDTA buffer (pH 5.0) and adding 0.03% (v/v) H₂O₂. Thereaction was stopped after 10 minutes by adding 100 μl of 1 M H₂SO₄.

The above-described ELISA assays performed on live Sf9 cells gave thefollowing results. FIG. 3 presents the data for live Sf9 cells infectedwith pAcB7 and pAcCD40 which were cultured for 48 hours in 24-wellplates. The antibodies used in the ELISA were: S2C6 (anti-CD40, openbars) and no primary antibody (hatched bars).

D. Host Animal Immunization

Female BALB/c mice were injected intraperitoneally at day 0 and day 14with 5×10⁶ Sf9 cells infected with AcCD40 virus, AcB7 virus or AcCd3virus (control virus). At day 21, 100 μl of serum was obtained to testfor the presence of specific antibodies. After a rest period of at leasttwo weeks, the mice received a final injection with 5×10⁶ cells infectedwith AcCD40 or AcB7 virus. Three days after this last injection, thespleen cells were used for cell fusion.

E. Generation of Hybridoma Clones

Splenocytes from immunized BALB/c mice were fused with SP2/0 murinemyeloma cells at a ratio of 10:1 using 50% polyethylene glycol aspreviously described by de Boer et al., J. Immunol. Meth. (1988)113:143. The fused cells were resuspended in complete IMDM mediumsupplemented with hypoxanthine (0.1 mM), aminopterin (0.01 mM),thymidine (0.016 mM) and 0.5 ng/ml hIL-6 (Genzyme, Cambridge, Mass.).The fused cells were then distributed between the wells of 96-welltissue culture plates, so that each well contained 1 growing hybridomaon average.

After 10-14 days the supernatants of the hybridoma populations werescreened for specific antibody production. For the screening of specificantibody production by the hybridoma clones, the supernatants of 12wells were pooled and used for fluorescent cell staining ofEBV-transformed B cells as described for the FACS Assay above.Subsequently, the supernatants of the positive pools were testedindividually. Positive hybridoma cells were cloned three times bylimiting dilution in IMDM/FBS containing 0.5 ng/ml hIL-6. Threehybridomas producing anti-CD40 antibodies are labelled 5D12, 3A8 and3C6. The data is presented in FIG. 4, which shows that a soluble form ofCD40, but not of B7 can block the binding of the anti-CD40 mAb 5D12 toCD40 expressing EBV-transformed B cells. FIG. 4 shows fluorescent cellstaining of ARC EBV-transformed B cells with 5D12 in the presence andabsence of soluble B7 and soluble CD40. 5D12 and the soluble B7, solubleCD40, or controls were preincubated at RT for 20 min before addition tothe ARC cells. FIG. 4A shows staing with 5D12 (doted line) or secondantibody only (solid line), FIG. 4B shows staining with 5D12 alone(dotted line) or preincubated with soluble B7 (solid line) and, FIG. 4Cshows stining with 5D12 alone (dotted line) or preincubated with solubleCD40.

Example 2 Costimulation of B-Cell Proliferation Using Anti-CD40 mAbs

Four hybridomas producing monoclonal antibodies against human CD40 weregenerated in Example 1. These mAbs were shown to bind to a similarproportion of tonsillar B cells as anti-CD40 mAb G28.5 does. de Boer etal. J. Immunol. Methods (1992) 152:15. Three of these monoclonalantibodies (5D12, 3A8 and 3C6) which were of the IgG2b subclass, weretested for their ability to deliver activation signals to human B cellsin the B-cell proliferation assay described above.

Human tonsillar B cells (4×10⁴ per well) were cultured in 200 μl inmicrowells in the presence of anti-IgM coupled to Sepharose beads (5μ/ml) (FIG. 5A) or in the presence of anti-IgM plus rIL-2 (100 U/ml)(FIG. 5B). Varying concentrations of the anti-CD40 mAbs S2C6, 5D12, 3C6or 3A8 were added and [³H]thymidine incorporation was measured at day 3after 18 h pulsing. Data presented in FIG. 5A are means derived fromexperiments with B-cell preparations from three different donors withduplicate incubations. Data of FIG. 5B are means of duplicateincubations from one experiment out of two with comparable results.

None of the novel anti-CD40 mAbs was able to significantly costimulatehuman B-cell proliferation in the presence of immobilized anti-IgM or inthe presence of immobilized anti-IgM and IL-2. In contrast, anti-CD40mAb S2C6 costimulated human B-cell proliferation in a concentrationdependent fashion.

Example 3 Induction of B-Cell Proliferation Using Anti-CD40 mAbs

The mAbs tested in Example 2 were tested for their ability to induceproliferation of human B cells in the Banchereau-like Assay describedabove, i.e., by presenting the anti-CD40 mAb on adherent cellsexpressing FcγRII. As antibody presenting cells, mouse 3T6 transfectantcells expressing the HR allellic form of human FcγRII were used. It wasobserved that anti-CD40 mAb S2C6 together with IL-4 induced substantialproliferation of tonsillar human B cells in this system, as assessed bymeasurement of [³H]thymidine incorporation. Anti-CD40 mabs 5D12, 3C6 or3A8 however, did not induce proliferation of human B cells in thisculture system (data not shown).

Example 4 Inhibition of S2C6 Stimulated B-Cell Proliferation UsingAnti-CD40 mAbs

The mAbs were also tested for their ability to inhibit the costimulationof human B-cell proliferation by anti-CD40 mAb S2C6 using the B-cellProliferation Assay described above. Human tonsillar B cells (4×10⁴ perwell) were cultured in 200 μl in microwells in the presence of anti-IgMcoupled to Sepharose beads (5 μg/ml) and anti-CD40 mAb S2C6 (1.25μg/ml). Varying concentrations of anti-CD40 mAbs 5D12, 3C6 or 3A8 wereadded and [³H]thymidine incorporation was assessed after 3 days. As acontrol anti-(glucocerebrosidase) mAb 8E4 was added in similarconcentrations. Barneveld et al. Eur. J. Biochem. (1983) 134:585. Dataare means±S.D. derived from experiments with B cells from two differentdonors with duplicate incubations.

It was found that each of the anti-CD40 mAbs 5D12, 3A8 and 3C6 couldinhibit the costimulation of anti-IgM induced human B-cell proliferationby mAb S2C6 (FIG. 6). In contrast, no significant inhibition was seenwith equivalent amounts of non-relevant mAb 8E4, directed toβ-glucocerebrosidase. Barneveld et al., supra. Thus, it was concludedthat these anti-CD40 mAbs do not deliver stimulatory signals to inducethe proliferation of human B cells, but, conversely, can inhibitstimulatory signals exerted by triggering CD40 with another mAb.Therefore, these mAbs were considered to be excellent tools toinvestigate whether signaling via CD40 plays a role in the stimulationof human B-cell proliferation by EL4B5 cells.

Example 5 Effects of Anti-CD40 mAbs on EL4B5-Induced Human B-CellProliferation

The effect of anti-CD40 mAbs on EL4B5-induced human B-cell proliferationwas tested using the B-cell Activation Assay described above. Humantonsillar B cells (1000 per well) were cultured together with irradiatedEL4B5 cells (50,000 per well) in the presence of 5% supernatant ofactivated human T cells and 5 ng/ml PMA. Anti-CD40 mAbs 5D12, 3C6 or 3A8were added in varying concentrations. As a control, mAb MOPC-141 (IgG2b)was added. After six days of culture, [³H]thymidine incorporation wasassessed.

FIG. 7 shows that addition of anti-CD40 mAbs 5D12, 3C6 or 3A8 resultedin a concentration-dependent inhibition of human B-cell proliferation.Data are means±S.D. derived from experiments with B cells from fourdifferent donors with duplicate incubations. [³H]-thymidineincorporation values found for incubations without mAb were (means±S.D.)10460±1843 cpm, 6982±1729 cpm, 4362±1020 cpm and 1543±3190 in the fourdifferent experiments, respectively. [3H]-thymidine incorporation in Bcells alone amounted to 40±5 cpm and in irradiated EL4B5 cells alone31±15 cpm.

Very potent inhibition occurred. At concentrations as low as 10 ng/mleach, the three anti-CD40 mAbs 5D12, 3C6 and 3A8 inhibited human B-cellproliferation completely. Half-maximal inhibition was found at about 1ng/ml. In contrast, isotype matched IgG2b mouse myeloma protein MOPC-141had no significant effect on [³H]-thymidine incorporation. Similarinhibition was observed when [³H]-thymidine incorporation was assessedat day 4 of the culture instead of day 6, thus excluding the possibilitythat the observed effect was due to a change in the kinetics of theproliferation under influence of the anti-CD40 mAb (data not shown).

For comparison, the influence of a few mAb directed against other B-cellsurface structures was investigated. Neither anti-CD20 mAb B1 or anti-B7mAb B7-24 (the latter mAb was generated by a prrcedure similar to thatused for generating the anti-CD40 mAb used in FIG. 7, in concentrationssimilar to those used in the experiments with the anti-CD40 mAb, had anyeffect on EL4B5-induced human B-cell proliferation (data not shown).Therefore, it may be concluded that the inhibitory effect of anti-CD40mAb on EL4B5-induced B-cell proliferation is not due to masking of theB-cell surface.

Example 6 Effects of hCD40.Hμ on EL4B5-Induced Human B-CellProliferation

In order to investigate whether EL4B5 cells expressed a membranestructure which binds CD40, a fusion protein consisting of theextracellular domain of CD40 and human IgM constant domains CH₂, CH₃ andCH₄ (hCD40.Hμ) was used for flow fluorocytometric analysis. Lane et al.,supra. Non-activated EL4B5 cells did not bind the fusion protein.However, upon culturing EL4B5 cells together with PMA (5 ng/ml) and 5%human T-cell supernatant, which are the conditions needed for activationof human B cells, a low binding of hCD40.Hμ was found (data not shown).This small shift in fluorescence was found consistently in threeindependent experiments. The minimal activation period needed forinduction of the CD40 binding was 24 hours. To determine whether bindingof hCD40.Hμ to the EL4B5 cells would inhibit EL4B5-induced human B-cellproliferation like anti-CD40 mAb did, the fusion protein was titratedinto cocultures of EL4B5 cells with human B cells using the B-cellActivation Assay described above. FIG. 8 shows that the fusion proteindid indeed inhibit [³H]-thymidine incorporation in aconcentration-dependent manner and, like the anti-CD40 mAb used in theexperiments shown in FIG. 7, was able to inhibit B-cell proliferationinduced by the EL4B5 cells completely.

Example 7 Effects of Anti-CD40 mAbs on Human T-Cell-Induced AntibodyProduction by Human B-Cells

The antibodies were also tested for their capacity to inhibitimmunoglobulin production by B cells, stimulated in a contact-dependentmanner with activated T cells using the T-cell helper assay describedabove. Human tonsillar B cells (10⁴/well) were cultured together withirradiated purified T cells (3000 rad, 10⁵/well) in 96-well plates,coated with anti-CD3 mAb and with or without different mAbs tocostimulate the T cells. After 8 days of culture the supernatants wereharvested for the determination of immunoglobulin production by the Bcells. Immunoglobulin production by the B cells was assessed by theELISA assay described above. Anti-CD40 mAb 5D12 was added in varyingconcentrations from the onset of the cultures. As a control, mnAbMOPC-141 was added. FIG. 9A shows that when T cells were stimulated withimmobilized anti-CD3 mAb and costimulated with soluble anti-CD2 andanti-CD28 mAbs, addition of anti-CD40 mAb 5D12 resulted in aconcentration dependent inhibition of IgG production by human B cells.IgM production by the B cells was inhibited to the same extent. Similarresults were obtained with the anti-CD40 mAbs 3C6 and 3A8 and with thehCD40.Hμ fusion protein.

The anti-CD40 mAbs of this invention exhibited very potent inhibition.At concentrations as low as approximately 30 ng/ml, each of the threeanti-CD40 mAbs gave 50% of maximal inhibition. In contrast, theisotype-matched IgG2b mouse myeloma protein MOPC-141 had no effect onthe immunoglobulin production.

The inhibitory effect by the three anti-CD40 mAbs was not specific forthe manner of activation of the T cells providing the CD40 ligand helperactivity. FIG. 9B shows that under all the T-cell stimulation conditions(anti-CD3 alone; anti-CD3+anti-CD2; anti-CD3+anti-CD28; andanti-CD3+anti-CD2+anti-CD28), addition of the anti-CD40 mAb 5D12 resultsin strong inhibition of immunoglobulin production by the human B cells.The inhibition is comparable to the amount of inhibition with thehCD40.Hμ fusion protein, known to completely block the CD40-CD40 ligandinteraction. The percentage of inhibition varied from 40 to 70%depending on the T-cell activation conditions. In contrast, theisotype-matched IgG2b mouse myeloma protein MOPC-141, or human IgM (ascontrol for the hCD40.Hμ fusion protein) had no effect on immunoglobulinproduction by the human B cells.

Deposition of Cultures

The hybridomas used in the above examples, to illustrate the method ofthe present invention were deposited in and accepted by the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, under the terms of the Budapest Treaty.

Hybridoma Deposit Date Accession No. B7-24 May 6, 1993 HB 11341 3C6 May6, 1993 HB 11340 5D12 May 6, 1993 HB 11339 3A8 January 30, 1996 HB 12024

The present invention has been described with reference to specificembodiments. However, this application is intended to cover thosechanges and substitutions which may be made by those skilled in the artwithout departing from the spirit and the scope of the appended claims.

7 50 base pairs nucleic acid single linear DNA (genomic) not provided 1GATCCACCAT GGAGCTCGAG ATCTAGAATT CTGCAGCCCG GGTACCGATC 50 27 base pairsnucleic acid single linear DNA (genomic) not provided 2 GCGCTGCAGCATCTGAAGCC ATGGGCC 27 27 base pairs nucleic acid single linear DNA(genomic) not provided 3 CGCGGTACCT TGCTTCTGCG GACACTG 27 61 base pairsnucleic acid single linear DNA (genomic) not provided misc_feature11..40 /note= “The underlining represents an epitope recognized by amonoclonal antibody used for affinity purification.” 4 GCGCGGTACCTTACTCCATG GGCATGTATT CCTCTTCCTC GTTATCAGGA AAATGCTGTT 60 G 61 32 basepairs nucleic acid single linear DNA (genomic) not provided 5 GCGTAGATCTGGTCTCACCT CGCCATGGTT CG 32 34 base pairs nucleic acid single linear DNA(genomic) not provided 6 GCGTGGTACC CCACACTCCT GGGTGGGTGC AGCC 34 61base pairs nucleic acid single linear DNA (genomic) not providedmisc_feature 11..40 /note= “The underlining represents an epitoperecognized by a monoclonal antibody used for affinity purification.” 7GCGTGGTACC TTACTCCATG GGCATGTATT CCTCTTCCTC ATCAGTCTTG TTTGTGCCTG 60 C61

We claim:
 1. A method for treating an antibody-mediated disease in apatient, said antibody-mediated disease being selected from the groupconsisting of systemic lupus erythemutosis (SLE), primary biliarycirrhosis (PBC), and idiopathic thrombo-cytopenic purpura (ITP), themethod comprising administering to a patient in need of such treatment atherapeutically effective amount of an anti-CD40 monoclonal antibody oran antigen binding fragment thereof, said monoclonal antibody orfragment thereof being free of significant agonistic activity andbinding to a human CD40 antigen located on the surface of a human Bcell, wherein the binding of the antibody to the CD40 antigen on thesurface of said B cell prevents the growth or differentiation of the Bcell.
 2. The method of claim 1 wherein the monoclonal antibody isselected from the group consisting of 5D12, 3A8 and 3C6, which aresecreted by a hybridoma having ATCC accession numbers HB 11339, HB 12024and HB 11340, respectively.
 3. The method of claim 2 wherein themonoclonal antibody is 5D12, which is secreted by a hybridoma havingATCC accession numbers HB
 11339. 4. The method of claim 2 wherein themonoclonal antibody is 3A8, which is secreted by a hybridoma having ATCCaccession numbers HB
 12024. 5. The method of claim 2 wherein themonoclonal antibody is 3C6, which is secreted by a hybridoma having ATCCaccession numbers HB
 11340. 6. The method of claim 1 wherein saidantibody-mediated disease is SLE.
 7. The method of claim 6 wherein themonoclonal antibody is selected from the group consisting of 5D12, 3A8and 3C6, which are secreted by a hybridoma having ATCC accession numbersHB 11339, HB 12024 and HB 11340, respectively.
 8. The method of claim 7wherein the monoclonal antibody is 5D12, which is secreted by ahybridoma having ATCC accession numbers HB
 11339. 9. The method of claim7 wherein the monoclonal antibody is 3A8, which is secreted by ahybridoma having ATCC accession numbers HB
 12024. 10. The method ofclaim 7 wherein the monoclonal antibody is 3C6, which is secreted by ahybridoma having ATCC accession numbers HB
 11340. 11. The method ofclaim 1 wherein the antibody-mediated disease is PBC.
 12. The method ofclaim 11 wherein the monoclonal antibody is selected from the groupconsisting of 5D12, 3A8 and 3C6, which are secreted by a hybridomahaving ATCC accession numbers HB 11339, HB 12024 and HB3 11340,respectively.
 13. The method of claim 12 wherein the monoclonal antibodyis 5D12, which is secreted by a hybridoma having ATCC accession numbersHB
 11339. 14. The method of claim 12 wherein the monoclonal antibody is3A8, which is secreted by a hybridoma having ATCC accession numbers HB12024.
 15. The method of claim 12 wherein the monoclonal antibody is3C6, which is secreted by a hybridoma having ATCC accession numbers HB11340.
 16. The method of claim 1 wherein the antibody-mediated diseaseis ITP.
 17. The method of claim 16 wherein the monoclonal antibody isselected from the group consisting of 5D12, 3A8 and 3C6, which aresecreted by a hybridoma having ATCC accession numbers HB 11339, HB 12024and HB 11340, respectively.
 18. The method of claim 17 wherein themonoclonal antibody is 5D12, which is secreted by a hybridoma havingATCC accession numbers HB
 11339. 19. The method of claim 17 wherein themonoclonal antibody is 3A8, which is secreted by a hybridoma having ATCCaccession numbers HB
 12024. 20. The method of claim 17 wherein themonoclonal antibody is 3C6, which is secreted by a hybridoma having ATCCaccession numbers HB 11340.